1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

449 Poster Developmental, Neurogenesis, and ConsumerResearchCANONICAL WNT SIGNALING DURING TASTE PAPILLAEFORMATIONIwatsuki K. 1 , Liu H. 2 , Mistretta C. 2 , Margolskee R.F. 1 1 Neuroscience,Mount Sinai School of Medicine, New York, NY; 2 School of Dentistry,University of Michigan, Ann Arbor, MITaste tissue development in mice is marked by the emergence of thetongue swelling around E11.5, followed by formation of the tongueplacode (E12.5), and taste papillae within the epithelium of the tongue(E13.5). The taste buds emerge at later stages, around the time of birth.As is the case with development of other epithelial tissues, formationand patterning of taste tissues are thought to be induced throughepithelial-mesenchymal interactions. Sonic hedgehog, bonemorphogenetic proteins and epidermal growth factor receptor areassociated with initiation and patterning of taste papillae. Othersignaling pathways, such as those involving the Wnts, essential in thedevelopment of many epithelial tissues, have not been examined for arole in taste tissue development. We have determined that specific Wntsignaling elements are expressed in developing taste tissue and thatcanonical Wnt signaling is associated with taste papilla formation.Topgal mice carry a beta-galactosidase (LacZ) reporter gene regulatedby the Tcf/Lef1-ß-catenin complex such that they can be used tomonitor canonical Wnt signaling pathways (DasGupta and Fuchs,1999). Using Topgal mice we observed that canonical Wnt signalingwas robustly activated during early stages of taste papilla formation, butless so during later stages, and was also active during taste buddevelopment. Work is in progress to identify the specific Wntsunderlying canonical signaling at various stages of taste papillae/buddevelopment. Supported by NIH DC003055 and DC003155 (RFM),DC00456 (CMM) and a JSPS fellowship (KI).450 Poster Developmental, Neurogenesis, and ConsumerResearchBMP-4 AND NOGGIN ALTER NEURON SURVIVAL ANDDIFFERENTIATION IN EMBRYONIC GENICULATE ANDTRIGEMINAL GANGLIA IN VITROMay O.L. 1 , Mistretta C.M. 1 1 School of Dentistry, University ofMichigan, Ann Arbor, MIBy rat embryonic day 16 (E16), geniculate and trigeminal ganglioncells innervate fungiform papillae and surrounding tongue epithelium,respectively, and thus are exposed to target-derived signaling factors,upon which they become dependent for survival and differentiation.Bone morphogenetic protein 4 (BMP-4), known to be involved inpatterning and regionalization of the nervous system, and its antagonist,noggin are expressed in tongue by E13 and dramatically influence tastepapilla development. To determine if these proteins not only regulateperipheral taste organs, but also influence development of neurons thatinnervate these targets, E16 geniculate and trigeminal ganglia wereexplanted and cultured with exogenous BMP-4, noggin, or brainderived neurotrophic factor (BDNF) for 3-6 days. Ganglia wereassessed for neuron survival and neurite outgrowth. Compared togeniculate ganglia exposed to BDNF, with exogenous BMP-4 or nogginthere was a substantial decrease in neuron number and reduced neuriteextension. Neuron reduction was especially pronounced with noggin.Furthermore, BMP-4 in particular induced neuron aggregation andneurite fasciculation. Although not as profound, neuron survival andneurite extension also were decreased in trigeminal ganglia exposed toeither BMP-4 or noggin. For both ganglia, addition of BDNF, BMP-4,and noggin together increased neuron survival relative to BDNF alone.We propose that these proteins, present in embryonic papillae, havevarying and balanced effects on ganglion survival and differentiation.Supported by NIDCD NIH grants DC00456 (CMM) and T32DC00011(OLM).451 Poster Developmental, Neurogenesis, and ConsumerResearchBDNF AND NT3 ATTRACT TRIGEMINAL NEURITESEgwiekhor A. 1 , Vatterott P. 1 , Rochlin M.W. 1 1 Biology, LoyolaUniversity of Chicago, Chicago, ILTrigeminal and geniculate axons are both attracted to gustatorypapillae, but are restricted to non-overlapping areas within theepithelium. We recently found that BDNF is an attractant for geniculateneurites. We therefore investigated which neurotrophins stimulatetrigeminal neurite growth by bath application and tested their ability toattract these neurites using slow release beads in collagen gels. Explantswere dissected from E15 and E18 rat embryos corresponding to in vivointralingual pathfinding and target penetration stages, respectively. Bathapplied NGF was the most potent and efficacious at eliciting outgrowthat E15 and E18. NT3 was more effective than BDNF at E15, but thisreversed at E18. NT3 stimulated finer fascicles than BDNF or NGF.Beads soaked in BDNF, NT3, and BDNF + NT3 did not promoteappreciable neurite growth from E15 ganglia, but BDNF- and NT3-soaked beads did attract E18 trigeminal neurites, as reflected byconvergence of proximal neurites toward the bead vs radial divergenceof distal neurites from the opposite side of the explant. BDNF + NT3beads elicited the most robust attraction. In preliminary experiments,NGF soaked beads biased outgrowth less than either BDNF or NT3 atE18. Taken together, our observations support the following model:NGF exerts the predominant trophic influence throughout pathfindingand targeting. BDNF and NT3 attract trigeminal axons to the papillaeepithelium, and NT3 promotes defasciculation within the epithelium.Supported by NIH R03 DC04965-01A1.452 Poster Developmental, Neurogenesis, and ConsumerResearchBDNF ATTRACTS GENICULATE NEURITES, NT4 DOESN'TRochlin M.W. 1 , Vatterott P. 1 , Egwiekhor A. 1 1 Biology, LoyolaUniversity of Chicago, Chicago, ILIn vivo studies raise the possibility that BDNF attracts geniculateaxons to gustatory papillae: Geniculate axons in mutant mice lackingBDNF or misexpressing BDNF exhibit aberrant intralingual trajectoriesand mistargeting, and BDNF mRNA is concentrated in the papillaeepithelium. However, it has not been determined if the guidanceinfluence of BDNF is direct, i.e., if BDNF is sufficient to attractgeniculate axons. To test this, in vitro studies are necessary. We coculturedcontrol or BDNF-soaked beads and geniculate gangliadissected from rat embryos at intralingual pathfinding stages (E14-16)and targeting stages (E17-18) in collagen gels. Several observationsargue that BDNF is not only trophic but tropic: Geniculate neuritesgrow exclusively (E15) or predominantly (E18) toward the beads, andneurites turn toward, stop at or encircle the beads. Furthermore, 25ng/ml BDNF in the bath did not block attraction to the BDNF soakedbead, suggesting a wide range over which BDNF gradients can besensed. Control beads soaked in media have no effect. Curiously, beadssoaked in NT4, which also signals through trkB, do not attractgeniculate neurites under these conditions. These data and those from invivo studies suggest a role for BDNF as an attractant for lingualgeniculate axons in vivo. We also recently demonstrated that tongueexplants promote and attract geniculate neurites, but the attractant isunlikely to be either BDNF or NT4 (Vilbig et al., J. Neurocytol.33:591). Future studies will assess the precise role and stages at whichBDNF acts as an attractant in vivo, and identify the explant-derivedattractant. Supported by NIH R03 DC04965-01A1.113